Nicotine is the primary constituent that regulates the maintenance of tobacco use. Many of the biological effects of nicotine arise directly or indirectly through the specific interaction with nicotinic cholinergic receptors. The nicotinic receptor family is composed of many diverse subtypes that display different anatomical locations, subunit compositions, and functional manifestations. Furthermore, chronic exposure of people (through smoking) or animals (through chronic nicotine administration) affects the number and function of nicotinic receptors in the brain. In addition, the effects of nicotine are influenced by the genetic makeup of the individual, either human or animal. However, several major questions remain to be addressed. This project will unite three research groups that have had a long-standing interest in nicotine or nicotinic receptors to investigate the role of nicotinic receptor subtypes in the regulation of response to nicotine after either acute or chronic exposure. Molecular biological, immunochemical, anatomical, biochemical, classical genetic, and pharmacological approaches will be used to study the biological bases of the heterogeneity of response to nicotine. Although it is known that chronic nicotine treatment increases the number of receptors measured by high affinity agonist binding, the precise molecular mechanism for this increase is unclear. Even less is known about those receptors that cannot be detected by ligand binding assays. With the interaction between the Collins group and the Lindstrom group, the effects of chronic nicotine treatment in vivo and in vitro on many nicotinic receptor subtypes can now be studied. The results of these experiments should clarify the role of these subtypes in tolerance development and clarify the molecular mechanisms underlying the changes in receptor levels observed with chronic treatment. Even though the numbers of nicotinic binding sites increase with chronic treatment, functional responses decrease. Because of the complexity of the nicotinic receptor system, a definitive assignment of the structure of the receptors mediating any of several biochemical measures has not been made. The interaction between the Collins group and the Heinemann group will begin to address this question. The determination of the effects of deleting a gene encoding a specific nicotinic receptor subunit on the behavioral, physiological and functional responses to nicotine will be of immense importance in assigning roles to each of the defined subunits. In addition, evaluation of the effects of gene deletion on tolerance development should further clarify the role of a given receptor subunit in the adaptive responses of the nervous system to chronic nicotine treatment. Furthermore, the application of immunological techniques to the knockout mice should provide useful information about the interaction among the remaining receptor subunits in the absence of one or more of the genes. The multidisciplinary approach achieved with this collaboration should expand understanding of the -molecular mechanisms underlying nicotine tolerance and dependence.